The production of bile is proportional to the net vectorial translocation of bile acids from the sinusoidal blood into the canalicular lumen. This requires the participation of specific bile acid transport mechanisms at both the sinusoidal and canalicular membranes. Moreover, bile acid transport across the canalicular membrane represents the rate limiting step in this physiological process. In order to understand the molecular basis of canalicular bile acid transport, we subjected rat liver canalicular membrane vesicles to bile acid affinity chromatography. An 110 kDa glycoprotein was purified and found to have many of the characteristics that might be expected of a canalicular bile acid transporter. In addition, the internal amino acid sequence was identical to another canalicular protein, previously characterized by cDNA cloning and functional studies as an ecto-ATPase. In recent publications I show that transfection of the ecto-ATPase cDNA into heterologous cells confers de novo synthesis of an 110 kDa glycoprotein, which is immunologically identical to the purified bile acid transport protein, and confers the capacity to pump out bile acids and the ability to hydrolyze ATP on these cells. Expression of the same cDNA after deletion of the carboxyl terminus by PCR-based deletion mutagenesis showed that the cytoplasmic tail of the canalicular bile acid transport/ecto-ATPase is essential for bile acid transport activity. These results therefore, demonstrate that bile acid efflux and ATP hydrolysis are two activities encoded by a single polypeptide. These results also demonstrate the development of a cell culture system (transfected COS cells), an assay for bile acid efflux in cultured cells, and the application of PCR-based mutagenesis techniques that will allow me to determine the structural basis for canalicular bile acid transport in this proposed project. Deletion mutagenesis has already been used to determine the relationship between ATP, ATPase activity and bile acid transport and to determine the structural requirements for tyrosine kinase-mediated and protein kinase C-mediated phosphorylation. We will now use the same approach to identify domains involved in bile acid binding and ATP binding. We will also examine the biogenesis, assembly and targeting of the transporter to the apical domain of polarized cells.
| Suchy, F J; Sippel, C J; Ananthanarayanan, M (1997) Bile acid transport across the hepatocyte canalicular membrane. FASEB J 11:199-205 |
| Sippel, C J; Dawson, P A; Shen, T et al. (1997) Reconstitution of bile acid transport in a heterologous cell by cotransfection of transporters for bile acid uptake and efflux. J Biol Chem 272:18290-7 |
| Sippel, C J; Shen, T; Perlmutter, D H (1996) Site-directed mutagenesis within an ectoplasmic ATPase consensus sequence abrogates the cell aggregating properties of the rat liver canalicular bile acid transporter/ecto-ATPase/cell CAM 105 and carcinoembryonic antigen. J Biol Chem 271:33095-104 |
